Parasitic flatworms (tapeworms and flukes) infect humans and livestock, causing a variety of diseases with pathologies that range from benign to deadly. These parasites have complex life cycles and remarkable capacity for growth, reproduction, longevity, and repair/regeneration. Despite their complexity and variations in their life cycles, parasitic flatworms share common features: perhaps most notably, they possess pluripotent adult somatic stem cells. Studies from free-living flatworms have shown that these stem cells (called neoblasts) are critical for normal tissue homeostasis, sexual reproduction, and regenerative capacity. Cytological studies revealed that parasitic flatworms maintain neoblasts, and recent molecular studies indicate that there are shared neoblast regulatory genes conserved among flatworms. Our long-term goal is to identify novel therapeutic targets against neoblasts that have the potential to treat multiple parasitic flatworm diseases. Our current objective is to identify and validate neoblast regulatory genes that are conserved among parasitic flatworms and that are necessary for parasite growth and/or reproduction. We have identified a suitable laboratory model (the rat intestinal tapeworm, Hymenolepis diminuta) that is amenable to gene discovery and functional validation in a high-throughput manner. We hypothesize that there are parasite-conserved neoblast regulatory genes critical for parasite growth and/or reproduction. To address this hypothesis, we propose the following two specific aims: (1) to identify genes that are conserved among parasitic flatworms and expressed in neoblasts; and (2) to functionally screen for regulators of parasite neoblasts that are required fo growth and/or reproduction. Neoblast-enriched genes will be identified using RNA-sequencing comparison of normal and neoblast-depleted H. diminuta. Candidate genes will be validated for co-expression in neoblasts by in situ hybridization. Using publically available genome and transcriptome assemblies of flatworms that infect humans and livestock, we will compile a candidate list of parasite-specific, neoblast-enriched genes. For functional validation, we will take advantage of our ability to culture H. diminuta in vitro. We have successfully established an in vitro regeneration assay and screening procedure to monitor tapeworm growth and development to reproductive maturity. We will establish methods to knock down expression of candidate genes from Aim 1 coupled to our in vitro regeneration assay to identify genes that are required for parasite growth and reproduction. These studies are innovative because we have designed a high-throughput and unbiased approach to uncover and functionally validate parasite-specific genes as regulators of parasite health, using H. diminuta as a model. The proposed work is significant, as we will identify high-confidence targets with the potential for broad efficacy against multiple parasite species. Together, our studies will fill important gaps in functional genomic studies of parasitic flatworms and have the potential to identify new therapeutic targets.